(Not Applicable)
Ligand-receptor assays or immunoassays are well-known in the art. Since their introduction in 1971, such assays have been utilized in a variety of applications to detect minute amounts of hormones, drugs, antibodies, and other substances suspected of being present in a given fluid sample. In this regard, researchers equipped with enzymes, antibodies, gene probes, and other reagents have made it possible to create chemical detection schemes for almost any compound of interest in a great diversity of applications. Among these applications are: commercial production of pharmaceuticals and food stuffs; food safety; diagnosis and treatment of disease in medical, veterinary, and agricultural environs; and detection and eradication of toxins in the environment. Common to all such applications is the requirement that chemical detection be performed in a timely, reliable, and cost effective manner.
Generally, bioassay schemes are developed and commercialized in formats suitable for use in laboratories equipped with general purpose instrumentation. Examples of these formats include immunoassay and DNA hybridization performed in test tubes, cuvettes, microtiter plates, columns, and electrophoretic gels. These formats usually include elaborate operational procedures and require frequent calibration using several calibrants which contain the analyte of interest at different concentrations. As a consequence, the high cost and complexity of operation associated with such formats limits widespread utilization thereof.
To address such drawbacks, developers and end users of immunoassays are increasingly replacing conventional bioassay formats which use test tubes, cuvettes, microtiter plates, columns, and electrophoretic gels with thin film chromatographic devices known as test strips. As is known in the art, the majority of test strips used for immunochemical detection of compounds are so called lateral flow test strips in which sample and reagents flow within the plane of the test strip. Advantageously, assays configured in a test stripformat can produce rapid results, are simpler to operate, and are more cost-effective than conventional formats. Additionally, such test strip assays may be utilized by unskilled laborers and can produce results on-site (i.e., outside a laboratory facility).
Generally, such assays rely on the binding of analytes by receptors to determine the concentration of such analytes in a given sample and are typically characterized as either competitive or non-competitive. Non-competitive assays generally utilize receptors in substantial excess over the concentration of analytes to be determined in the assay. Typical of such non-competitive immunoassays include sandwich assays, which detect the presence of an analyte by binding two receptors thereto. In such arrangement, the first receptor, which is typically an antibody is bound to a solid phase such that when the analyte is present, such analyte becomes affixed thereto. A second receptor having a label covalently attached thereto, which may comprise a radioactive, fluorescent, enzymatic, dye or other detectable moiety (collectively referred to as tracers), is introduced to the assay which consequently binds to the bound ligand, to the extent the ligand is present, and thereafter produces a signal consistent with the presence of such ligand. If the sample does not contain the molecules of interest, the labeled receptor is carried past the immobilized receptor without reacting which, as a consequence, will not cause a change in the membrane. Such non-competitive immunoassays are primarily useful for the detection of large molecules such as proteins, large hormones or molecules which have multiple binding sites, such as human chorionic gonadotropin (HCG) and typically will not work with small molecules that have only one binding site.
Competitive assays, in contrast, generally involve competition between a ligand present in a given sample, and a ligand analog having a tracer/label covalently linked thereto to permit detection for a limited number of binding sites provided by the ligand receptor, which typically comprises an-antibody bound to a solid phase. Such assays are particularly suited to detect smaller molecules, such as drugs and drug metabolites. In this context, drug analogs are utilized that have been covalently bound to a protein which is then immobilized on a membrane. Antibody specific to the drug is then labeled and immobilized on a porous pad. When a sample is added which is suspected of containing a given analyte, such sample dissolves the labeled antibody and carries it into contact with the immobilized drug-protein region. If there is little or no drug in the sample, a large amount of the labeled antibody is bound to the immobilized drug-protein region which, consequently, produces a detectable signal. If the sample contains a high amount of drug, little or no labeled antibody is bound to the immobilized drug-protein region and thus in turn gives little or no signal.
Today, rapid immunoassays generally consists of an adhesive-covered plastic backing onto which several porous pads and a piece of protein-binding membrane are attached. The membrane typically contains a section that has been impregnated with a binding partner (i.e., a receptor or ligand analog). A second pad is typically provided which contains a labeled target molecule or labeled antibody protein-binding membrane. When a sample suspected of containing a target ligand is contacted with the immunoassay, such sample dissolves the labeled element or tracer and the capillary action of the protein-binding membrane subsequently draws the sample with tracer dissolved therein into contact with the impregnated binding partner. When this reaction occurs, there is a change in the appearance of the binding membrane, with the difference providing a qualitative indication of the presence or absence of the ligand suspected of being present in such sample.
Typical examples of this form of test strip are those which visually display two parallel lines (known as capture lines) on a test membrane. Capture lines consist of immobilized capture reagents or receptors which are preapplied to the test membrane during its manufacture. In this regard, both virtually all prior art assays, whether competitive or non-competitive, typically deploy a receptor immobilized on a membrane, as assessed above. A schematic representation of the construction of a typical lateral flow test strip is as follows:
reagent pad//test membrane capture line test membrane/capture line/test membrane//absorbent pad.
where:
symbol/designates a phase boundary within a single chromatographic medium; and
symbol//designates a union of two separate mediums (chromatographic or other medium).
One of the two capture lines serves as an indication that the test strip performance has not been compromised. In this regard, such capture line serves an important function by providing quality assurance and integrity of the assay, which is generally considered necessary insofar as individual test strip performance can vary greatly. The second of such capture lines becomes visible only when the sample contains an amount of analyte in excess of a minimum concentration (threshold concentration). Exemplary of such so prior art systems and methodologies include the immunoassay systems and test strips disclosed in U.S. Pat. No. 5,658,723, issued on Aug. 19, 1997, to Oberhardt entitled xe2x80x9cImmunoassay System Using Forced Convection Currentsxe2x80x9d and U.S. Pat. No. 5,712;170, issued on Jan. 27, 1998, to Kouvonen, et al. entitled xe2x80x9cTest Strip, Its Production and Usexe2x80x9d, the teachings of each of which are expressly incorporated herein by reference.
Unfortunately, despite their cost-effectiveness and simplicity of use, typical test strip format assays are less accurate, less precise, and less sensitive to analyte presence than conventional formats. As a result of such drawbacks, the application of test strip format assays has been limited to semi-quantitative or qualitative assays. Among the more significant factors that contribute to the inaccuracy and imprecision of test strip format assays include the manufacture and use of capture lines. As is widely recognized, the manufacture of consistently uniform capture lines requires elaborate material control and manufacturing processes with rigid specifications that must operate within narrow tolerances. Moreover, to function properly, most test strip formats require that the analytes to be detected must be uniformly captured in a precise geometry at a precise location on the test strip and that factors such as the ambient humidity present at the time of test strip manufacture, type of membrane utilized in such manufacturing process, and a capture reagent-receptor itself contributing greatly to assay inaccuracies and false readings. A detailed discussion regarding the drawbacks associated with the binding of protein capture reagents in immunochromatographic assays can be found in Jones, Kevin D., xe2x80x9cTroubleshooting Protein Binding in Nitrocellulose Membranesxe2x80x9d, Part I, IVD Technology, Volume V, No. II, March-April 1999, pages 32-41 and Part II, IVD Technology, Volume V, No. III, May-June 1999, pages 26-35, the teachings of which are expressly incorporated herein by reference.
Of further significant disadvantage is the fact that virtually all test strip format assays are formed to have a sequential, generally-linear configuration so as to facilitate the necessary lateral flow thereacross. Due to the fact that such fluid sample must necessarily migrate from its starting point across the reagent pad, the test membrane, and ultimately across the capture line(s) for detecting the presence of the suspect analyte, a substantial portion of the target analyte is often caused to become dispersed or otherwise inhibited from reaching the bound receptors forming the capture line. As such, a substantial portion of the target analyte sought to be detected can and frequently is missed altogether which can adversely effect the quantitative and qualitative results generated by such assays.
Such potential to inadvertently fail to detect the presence of a target analyte, whether it be through losing the target analyte sought to be detected or simply overlooking its presence is particularly problematic when attempting to detect the presence of cancer cells in a given fluid sample. For example, in a slingle 10 ml tube of blood, there are approximately fifty billion cells, and the presence of so much as one cancer cell among this cell population can be indicative of the presence of micrometastasis. Utilizing conventional screening techniques, such blood samples are typically processed to isolate the leukocytes present in such sample, which advantageously reduces such fluid sample for example, from 10 ml to between 1.0 to 0.5 ml, which consequently reduces the cell population from approximately fifty billion to approximately one hundred twenty million. Such procedure, however,typically results in the loss of cancer cells and as such may inadvertantly remove the cancer cells sought to be detected.
In order to screen for cancer cells, such resultant sample is then portioned and applied to many microscope slides at a ratio of approximately one million cells to each slide, via well-known procedures such as cytospin. As is known, each slide prepared represents another inadvertent loss of cancer cells. Each respective one of the slides must then be meticulously scanned using a microscope. In this respect, each slide is typically divided into four hundred magnified fields comprised of one square millimeter areas that are each reviewed. Typically, such scanning process takes on average a half hour per slide. As a result, to thoroughly examine the condensed population of one hundred twenty million leukocytes present in one 10 ml blood sample requires the examination of one hundred twenty slides, producing forty eight thousand images that must be examined over a sixty hour period. Accordingly, even to the extent prior art assay techniques are effective at labeling a target cell sought to be detected, the process by which such labeled cell is ultimately isolated and detected is inherently unreliable, tedious and time consuming.
It is therefore desirable to devise an alternative lateral flow device which can capture analyte at a precise location, and preferably at the starting point or point of contact at which the fluid sample is deposited. It would likewise be desirable to devise an alternative assay that can capture an analyte at such starting point or point of contact in a precise geometry without the use of preapplied capture lines. There is also a need for an assay that has greater sensitivity in reproduceability than prior art assays and methods and is likewise inexpensive, less labor intensive, relatively easy to manufacture, and capable of being utilized for a wide variety of applications. There is further a needs for an essay that may capture prepared cell specimens directly onto microscopic slides and significantly reduce the time and number of required images to be produced therefrom, particularly with respect to the isolation and detection of cancer cells.
The present invention specifically addresses and alleviates to the above-identified deficiencies in the art. In this regard, the present invention pertains to several novel bioassay methodologies, chromatographic devices, and an optional multimode photometer/analyzer which together can perform bioassays with accuracy and precision like that of conventional laboratory formats while retaining the operational simplicity, rapid analysis, and cost-effectiveness like that of test strip formats. The chrcmatographic devices and novel bioassay methodologies of the present invention further minimize problems associated with the manufacture of test strips which incorporate preapplied capture lines and further, can enable an analyte to be detected in a fluid sample in a manner that efficiently conserves and isolates the analyte present in such sample. Moreover, multimode photometers, novel test strip devices, and unique chemical analysis methods of the present invention represent a versatile, cost effective, simple, and accurate system which can quantify the amount of a chemical substance present in a sample that has not heretofore been available via prior art bioassay test strips. According to a first aspect of the present invention, there is provided a novel magnetic chromatography method which consists of the steps of contacting activated magnetic particles suspended in a reaction mixture with a chromatographic medium (e.g., test strip or chromatographic plate), and thereafter applying a magnetic field thereto. As the activated magnetic particles flow laterally within the plane of the medium they encounter the applied magnetic field. The applied magnetic field attracts the magnetic particles forming a magnetic barrier that selectively retains magnetic particles while allowing the reaction mixture to continue to flow laterally thereacross. In a more highly preferred embodiment, which is particularly useful in performing cell capture assays, the reaction mixture with magnetic particles is contacted upon an intermediate portion of a chromatographic medium having a magnetic field applied at the point of contact. The reaction mixture is thus caused to flow bilaterally across the medium, with the magnetic particles having the analyte of interest complexed therewith being captured at the starting point, or point of sample introduction, thus conserving the amount of analyte in the reaction mixture that would otherwise become lost through reaction mixture flow.
As such, there is thus eliminated the conventional capture lines formed by bound receptors that are utilized in prior art immunoassays as well as analyte loss that can occur during the assay. In this regard, a capture line is in effect assembled during the assay, and preferably at the outset. Advantageously, the magnetic chromatography assay methods of the present invention allow test strips and the like to be manufactured without preapplied capture lines. However, the methods of the present invention also anticipate a magnetic chromatography test strip having both preapplied capture lines and capture lines formed during the bioassay using magnetic chromatography as may be desired for a specific application.
The novel methods of the present invention may further deploy one or more applied magnetic field source(s) applied to the chromatography test strip assembly to detect multiple spectrophotometric analysis. For example, a common bar magnet or magnetic strip can be attached to the test strip backing with adhesive at one or more locations. Alternately, the magnetic source can be external to the test strip assembly whereby the magnetic source is selectively positioned in close proximity with the test strip while magnetic particles flow laterally therewithin. In preferred embodiments of the present invention, the source of the applied magnetic field may comprise either permanent magnets or electromagnets.
The present invention further includes a novel magnetic chromatography test strip for performing a bioassay that conserves the amount of analyte sought to be detected in a reaction mixture by forming a capture line or zone at the point at which the reaction mixture is contacted with the test strip. According to a preferred embodiment, the test strip comprises an elongate backing having first and second ends and an intermediate portion disposed therebetween. Upon the intermediate portion are a first vertical mesh for facilitating vertical flow and second lateral mesh for facilitation lateral flow, the latter being disposed underneath the first mesh. On opposed sides of the lateral flow mesh are absorbent pads that, in use, cause the reaction mixture ultimately passing through the first and second meshes to flow bilaterally. Such strip further preferably includes at least one magnet that is positioned underneath the vertical flow mesh and second lateral flow mesh.
In use, a reaction mixture is sequentially caused to flow from the vertical flow mesh, to the lateral flow mesh, and ultimately to the first and second absorbent pads disposed on the opposed sides of the lateral flow mesh. Preferably, such reaction mixture is deposited upon the strip by means of a sample well. The applied magnetic field provided by the magnet attracts the magnetic particles having the analyte of interest complexed thereto, which thus causes the same to be selectively retained at the site of deposition while the remainder of the reaction mixture continues to flow thereacross and ultimately to the respective of the absorbent pads. Such test strips are particularly well suited for the detection and isolation of cells, such as cancer cells, bacteria and the like, which are otherwise difficult to identify and isolate through priorart methods.
There is further provided as part of the present invention a novel analyzer comprised of a multimtode photometer which can measure front surface fluorescence, luminescence and reflectance at a single focal point on the test strips of the present invention. According to a preferred embodiment, the multimode photometer consists of a base and optical canopy which collectively define an optical tunnel into which at least one test strip may be disposed. The chamber may include a magnetic source or be designed to be placed in close proximity to a magnetic source such that the test strip having activated magnetic particles flowing laterally therewithin may be caused to become substantially bound at a specific site or sites upon the test strip. When so arranged, a light or radiation source may be focused upon the test strip disposed within the optical tunnel such that the light or radiation may be aligned with the magnetic source and the reflected or emitted light from the test strip analyzed for analyte presence. Light and radiation of differing wave lengths may be utilized to determine the presence of appropriate analytes as per conventional spectrophotometric analysis. Optical filters and photodetectors may further be deployed as may be necessary for a particular spectrophotometric applications.
It is therefore an object of the present invention to provide a novel magnetic chromatography assay and method utilizing a test strip format that has greater sensitivity and reproduceability than prior art test strip assays.
Another object of the present invention is to provide a novel magnetic chromatography assay and method that utilizes a test strip format but dispensesr with a need to form a capture line by binding receptors to a test membrane.
Another object of the present invention is to provide a novel magnetic chromatography assay and method that can be arrayed in a test strip format and utilized to provide quantitative analysis.
Another object of the present invention is to provide a novel magnetic chromatography assay and method that can form a capture line at the point of contact. where a reaction member comes into contact with such test strip.
Another object of the present invention is to provide a novel magnetic chromatography assay and method that can conserve the amount of analyte present in a reaction mixture by identifying and isolating the same at the initiation of the performance of such assay.
Another object of the present invention is to provide a novel magnetic chromatography assay and method that may be adapted to provide quantitative and qualitative analysis for multiple analytes.
Another object of the present invention is to provide a novel magnetic chromatography assay and method that is easy to use, of simple construction, and inexpensive to manufacture.
Another object of the present invention is to provide a novel magnetic chromatography assay and method that may be utilized to provide spectrophotonmetric analysis, including but not limited to, surface reflectance, surface fluorescence, and surface luminescence.
Another object of the present invention is to provide a novel magnetic chromatography assay and method which may be configured to isolate target cells and facilitate the ability to detect the same.
Another object of the present invention is to provide a novel magnetic chromatography assay and method which may be adapted to capture cells directly onto slides and facilitate microscopic examination thereof.
Another object of the present invention is to provide a novel magnetic chromatography assay and method which may be configured to perform individual sample analysis, batch sample analysis, and linear-array analysis.
Another object of the present invention is to provide a novel magnetic chromatography assay and method wherein such assay may be configured to be reusable or disposable.
Another object of the present invention is to provide a novel magnetic chromatography assay and method which will accommodate conventional reagents prepackaged in unit. doses.
Another object of the present invention is to provide a novel magnetic chromatography assay and method that can be used for quantitative, semi-quantitative, and qualitative immunoassay of analytes and DNA hybridization assays.
Another object of the present invention is to provide an optical analyzer consisting of a multimode photometer for performing spectrophotometric analysis, including but not limited to, surface reflectance, surface fluorescence, and surface luminescence.
Another object of the present invention is to provide an analyzer consisting of a multimode photometer which is of simple construction, easy to utilize, and may be configured to perform individual sample analysis, batch sample analysis, and linear-array analysis.
Another object of the present invention is to provide an analyzer consisting of a multimode photometer that, when utilized in conjunction with the magnetic chromatography assays of the present invention, may be utilized to quantitate the amount of a given analyte at a fixed location on a test strip assay, irrespective of orientation of such assay and lateral flow of reaction mixture utilized therewith.